Conjugation of molecular entities to other molecules or materials is used in formulation of a wide variety of compounds or materials used for medicine, biology, industrial processes, etc. (Hayashi and Ikada, 1991, Petach et al., 1994, and Krajwska et al., 1990). For many applications, particularly in the biomedical areas, formation of well-defined covalent linkages between two compounds or substances must be performed in aqueous solutions (Brewster et al., 1989, Schubiger et al., 1996, Duchene et al., 1987).
Many reaction pathways are being used to form specific covalent bonds between compounds (Duchene et al., 1987, Annuziato et al., 1993, and Liu et al., 1996). One of the most common approaches is to react an active functional group with amine groups in a highly specific manner (Schubiger et al., 1996, Lewis et al., 1994). For example, conjugation of monoclonal antibodies or other proteins in aqueous solution with a spectrum of molecular species including, for example, drugs, toxins, fluorescent molecules, and metal chelates, is frequently effected through the formation of a covalent bond. For example, the bond can be amide or imidate bonds, with amine group(s) on the biomolecule (Duchene et al., 1987, Annuziato et al., 1993, Liu et al., 1996, Lewis et al., 1994, Moi et al., 1988, Cox et al., 1990, Kline et al., 1991).
The use of HMP group(s) to conjugate or cross-link one molecule to another molecule or substance containing one or more amine groups provides a novel alternative to other methods currently in use to produce well-defined conjugates. It is known that --CH.sub.2 OH sidearms on tris (hydroxymethyl) phosphine (THP) react with amines by Mannich-type condensation reactions with amine groups in aqueous solutions giving aminomethylphosphines (Angiolini, 1990). THP, the most simple HMP ligand, has recently been used to immobilize enzymes (Hayashi and Ikada, 1991, Petach et al., 1994). In that study, THP was mixed with a resin, in bulk, for one hour. The resin was washed in a solution containing urease enzyme and, after incubation for six hours, the immobilized urease was rinsed several times with buffer (Petach et al., 1994). While this application with THP demonstrated the feasibility of using this simple ligand as an immobilization agent, the immobilized products produced were not defined and, undoubtedly, contained a mixture of products including polymers and other compounds possessing undefined aminomethylphosphines linkages. In addition, the effect of pH on both reaction yields and/or product distribution of this method is unknown. Because that study was performed in bulk using non-stoichiometric quantities of reagents, the immobilization yields appear to be low, particularly since no estimates of reaction yields were provided (Petach et al., 1994).
In order for HMP groups to be used as effective functionalities for conjugation or cross-linking one molecule to another compound or substance, their reactions with amines must 1) result in formation of well-defined covalent linkages and 2) form that linkage in high yields. Furthermore, it would also be desirable that the conjugation or cross-linking reactions be conducted under relatively mild conditions over a wide pH range in aqueous solutions, particularly for those involving biomolecules.
Even though THP is known to react with amines via a Mannich-type condensation, the studies to determine the yields, rates of reactions, and conditions required to produce a specific type bonding between HMP groups and amines have, to date, not been performed. For example, it is assumed that the only bond formed when hydroxymethylphosphines react with primary amines is P--CH.sub.2 --N(H)--linkage (Petach et al., 1994). In addition, the HMP containing molecule THP is expected to react with amine groups to produce polymeric products (Reaves and Guthrie, 1956).
In contrast, applicants have demonstrated, as set forth below, that HMP-containing compounds containing two HMP groups separated by two carbon atoms can react with primary amines in aqueous solutions to form a cyclical linkage (i.e., --PN--). Further, applicants have found that HMP groups react efficiently with amines over a wide pH range (i.e., pH 2-9) and that a specific linkage can be formed in high yields. Applicants have also demonstrated that HMP groups can react with secondary amine groups under similar conditions where the linear P--CH.sub.2 --N(R) linkage (where R.noteq.H) is formed and in high yields.
The results of the studies reported herein demonstrate that HMP groups, under appropriate reaction conditions, can form a specific aminomethyl-phosphorus bond in high yields with amine groups in aqueous solutions. These results provide evidence that HMP reaction(s) with amines can be used as a vehicle for conjugation or cross-linking one compound with another compound or substance in aqueous media over a wide pH range (i.e., pH 2-9). The resulting conjugated or cross-linked product will be a well-defined chemical entity.
Until recently, very few compounds of this type have been synthesized (Hayashi and Ikada, 1991, Petach et al., 1994, Frank and Drake, 1972). A major reason for paucity of RP(CH.sub.2 OH).sub.2 compounds relates to the difficulties in their synthesis. Since these types of compounds have not been available, characterization of HMP group reactions with amines has not been fully elucidated. Recent efforts by applicants to develop strategies for the synthesis of a variety of HMP-based compounds have been successful. With the availability of new HMP-containing compounds, applicants have been able to study the HMP-amine reactions in aqueous solutions under various conditions, including variations in pH. Furthermore, the new synthetic methods developed by applicants have provided evidence that it is possible to synthesize and chemically characterize a wide variety of compounds containing one or more HMP functionalities attached at a predetermined position on the molecule. These fully characterized molecules can, be used to conjugate or cross-link one molecule containing a HMP group to another molecule or material possessing primary or secondary amine group(s) located at specific site or sites on the molecule.
Therefore, it would be both desirable and advantageous to have a method for conjugating or cross-linking one molecule to another compound or substance which results in the formation of well-defined covalent linkages in high yields. Furthermore, it would also be desirable that the conjugation and/or cross-linking reactions be conducted under relatively mild conditions over a wide pH range in aqueous solutions, particularly for those involving biomolecules.